Omar Alomair Homod Alherbi Fisal Alkulify DNA Sequencing Omar Alomair Homod Alherbi Fisal Alkulify

Introduction: The term DNA sequencing encompasses biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide.

Introduction: The sequence of DNA constitutes the heritable genetic information in nuclei, plasmids, mitochondria, and chloroplasts that forms the basis for the developmental programs of all living organisms.

Introduction: Determining the DNA sequence is therefore useful in basic research studying fundamental biological processes, as well as in applied fields such as diagnostic or forensic research.

Introduction: The advent of DNA sequencing has significantly accelerated biological research and discovery. The rapid speed of sequencing attainable with modern DNA sequencing technology has been instrumental in the large-scale sequencing of the human genome.

How do we Sequence DNA First you need to know a few key terms: Plasmid A 'plasmid' is a small, circular piece of DNA that is often found in bacteria. This innocuous molecule might help the bacteria survive in the presence of an antibiotic, for example, due to the genes it carries.

Key terms Genome In biology the genome of an organism is its whole hereditary information and is encoded in the DNA (or, for some viruses, RNA). This includes both the genes and the non-coding sequences of the DNA

Key terms Plasmid To scientists, however, plasmids are important because (i) we can isolate them in large quantities, (ii) we can cut and splice them, adding whatever DNA we choose, (iii) we can put them back into bacteria, where they'll replicate along with the bacteria's own DNA

Key terms BAC The term 'BAC" is an acronym for 'Bacterial Artificial Chromosome', and in principle, it is used like a plasmid. We construct BACs that carry DNA from humans or mice or wherever, and we insert the BAC into a host bacterium.

Key terms BAC As with the plasmid, when we grow that bacterium, we replicate the BAC as well. Huge pieces of DNA can be easily replicated using BACs Using BACs.

Key terms Primer A primer is a strand of nucleic acid that serves as a starting point for DNA replication.

Key terms Vector The 'vector' is generally the basic type of DNA molecule used to replicate your DNA, like a plasmid or a BAC .

Key terms Insert The 'insert' is a piece of DNA we have purposely put into another (a 'vector') so that we can replicate it.

DNA Sequencing DNA PREPARATION DNA needs to be purified from cells. First, the cells and their nuclei are broken open. This can be accomplished by mechanical methods, such as grinding, or by chemical methods that break apart cell membranes. The DNA floating around in this “soup” is still coated with protective proteins. The DNA can be selectively removed from this soup by precipitating it and DNA-binding proteins can be cleaned away.

Automated DNA Sequencing DNA PREPARATION Very large pieces of DNA, such as whole chromosomes or genomes, are cut into smaller pieces and stored in vectors (plasmids), which are larger pieces of DNA with the ability to be reproduced when placed in host cells such as bacteria.

Automated DNA Sequencing DNA PREPARATION Bacteria containing a vector are placed in culture medium, where they multiply a million-fold or more. Each time a bacterium divides, the DNA vector placed inside is also copied. In this way, the target DNA can be multiplied exponentially. Each of the copied DNA pieces is called a clone.

Automated DNA Sequencing SEQUENCING REACTION The sequencing reaction itself consists of four steps, which will be covered in detail in this section. First, the double-stranded DNA is separated into single strands, and a small starter piece of DNA called a primer binds to one of the strands, called the template strand.

Automated DNA Sequencing SEQUENCING REACTION In the extension step, a new DNA strand is made that is complementary to the template strand. Starting at the primer, DNA polymerase uses the template strand as a guide to recreate the second DNA strand.

Automated DNA Sequencing SEQUENCING REACTION The termination step is the key to the sequencing reaction. Strand extension is halted by the incorporation of a dye-labeled terminator nucleotide, which identifies the base at the position where strand extension stopped. When many strand termination reactions are performed together, each of the bases in a DNA strand can be identified.

Automated DNA Sequencing STRAND SEPARATION Double-stranded DNA needs to be denatured, or separated into single strands, before it can be sequenced. This process is accomplished by heating the DNA, which disrupts the hydrogen bonds and VanDerWaals forces that hold the two chains of DNA together in a double helix.

Automated DNA Sequencing PRIMER ANNEALING A small single-stranded DNA piece of about 20 bases, called an oligonucleotide, is annealed to the denatured template strand. This oligonucleotide is needed to “prime” the next step, DNA extension.

Automated DNA Sequencing PRIMER ANNEALING the two DNA strands that were separated in the preceding step could just snap back together. This is avoided by using a rapid cooling process, which gives the small nucleotides an advantage over long DNA strands in annealing. In addition, a large excess of primers is used to again ensure that the primers will out-compete the complementary DNA strand for annealing to the template.

Automated DNA Sequencing PRIMER ANNEALING The oligonucleotide primer must be of complementary sequence to the template strand in order to bind by base-pair interactions.

Automated DNA Sequencing PRIMER EXTENSION During the extension phase, a bacterial DNA polymerase enzyme begins assembling a new DNA chain from the individual nucleotide building blocks, or dNTPs, provided in the reaction mixture.

Automated DNA Sequencing PRIMER EXTENSION The nucleotides are added in the order specified by the complementary bases in the template strand. DNA polymerase cannot start copying a template strand without a small piece of DNA to start the extension process. This is why the primer was added in the previous step.

Automated DNA Sequencing CHAIN TERMINATION The reaction mixture also contains small amounts of each of the 4 dideoxynucleotides, or “ddNTPs,” which lack the 3'-hydroxyl group necessary for chain extension. Whenever a dideoxynucleotide is incorporated into a growing DNA chain, it terminates chain growth because another nucleotide cannot be attached to it. Each of the four ddNTPs is labeled with a different dye, which can later be detected using a special laser.

Automated DNA Sequencing CHAIN TERMINATION The DNA polymerase occasionally incorporates a labeled dideoxynucleotide into a growing DNA strand. This doesn’t happen very often, because the concentration of dideoxynucleotides is much lower than the concentration of dNTPs in the reaction mixture. The ratio of dNTPs to ddNTPs is carefully balanced to get just the right number of chain termination events.

Automated DNA Sequencing PUTTING IT ALL TOGETHER An actual sequencing reaction mixture contains thousands of DNA template strands, which are all being sequenced simultaneously. Simply by chance, some annealed primers will only be extended a few nucleotides before the chain extension is terminated by the addition of a ddNTP.

Automated DNA Sequencing PUTTING IT ALL TOGETHER However, other primers will form a long chain of DNA before a ddNTP is incorporated. Thus, there will be a population of DNA strands in the reaction, some very short, some very long, and every possible length in between.

Automated DNA Sequencing PUTTING IT ALL TOGETHER To further increase the yield of sequenced strands, the sequencing reaction is performed in a thermal cycler, which cycles through the heating and cooling steps dozens of times, in effect repeating the sequencing reaction many times in one experiment.

Automated DNA Sequencing CAPILLARY ELECTROPHORESIS The newly synthesized DNA strands, each labeled with one of four dyes, are now sorted by length using capillary electrophoresis. First, the reaction mixture is heated to keep the newly synthesized single strands from annealing with the template DNA strand. The dye-labeled single strands are loaded onto a tiny capillary tube containing a viscous, gel-like material.

Automated DNA Sequencing CAPILLARY ELECTROPHORESIS An electrical current pulls the negatively charged DNA strands through the capillary. This tube is not much thicker than a human hair and is 1 to 3 feet long, sufficient to separate strands that differ in length by only one base. Because of the small dimensions involved, preparation of the capillary and loading of the sample are computer controlled.

Automated DNA Sequencing CAPILLARY ELECTROPHORESIS Shorter DNA strands migrate through the gel material more quickly, and come out the bottom of the capillary first, while longer strands become tangled in the gel material and take longer to emerge out the bottom.

Automated DNA Sequencing CAPILLARY ELECTROPHORESIS As the strands emerge out the bottom of the capillary they pass through a laser beam that excites the fluorescent dye attached to the dideoxynucleotide at the end of each strand. This causes the dye to fluoresce, or glow, at a specific wavelength, or color. This color is then detected by a photocell, which feeds the information to the computer.

Automated DNA Sequencing COMPUTER ANALYSIS The computer displays the information received from the photocell as an electropherogram, which is a tracing of signal received by the photodetector in each of the four wavelengths.

Automated DNA Sequencing

In case of manual DNA sequencing The DNA sample is divided into four separate sequencing reactions containing the four standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase.

In case of manual DNA sequencing The DNA sample is divided into four separate sequencing reactions containing the four standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase.

In case of manual DNA sequencing To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP). These dideoxynucleotides are the chain-terminating nucleotides, lacking a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides during DNA strand elongation. Various DNA fragments of varying length are created in the four separate sequencing reaction .

In case of manual DNA sequencing - The newly synthesized and labeled DNA fragments are heat denatured, and separated by size (with a resolution of just one nucleotide) by gel electrophoresis on a denaturing polyacrylamide-urea gel. - Each of the four DNA synthesis reactions is run in one of four individual lanes (lanes A, T, G, C); the DNA bands are then visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel image. (A dark band in a lane indicates a DNA fragment that is the result of chain termination after incorporation of one particular dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP). - The relative positions of the different bands among the four lanes are then used to read (from bottom to top) the DNA sequence as indicated.

Automated VS manual radioactivity is not used, Automated DNA sequencing has the following advantages over manual DNA sequencing radioactivity is not used, gel processing after electrophoresis and autoradiography are not needed, the tedious manual reading of gels is not required as data are processed in a computer, the sequence data is directly fed into and stored in a computer, the separation of the same reaction products can be repeated to recheck the results in cases of doubt since they can be stored for a long period of time, and it is extremely fast.

Application Of DNA Sequencing Human Genome Project Primary Application of DNA Sequencing is Human Genome Project Which is an international scientific research project with a primary goal to determine the sequence of chemical base pairs which make up DNA and to identify the approximately 25,000 genes of the human genome from both a physical and functional standpoint. The work on interpretation of genome data is still in its initial stages. It is anticipated that detailed knowledge of the human genome will provide new avenues for advances in medicine and biotechnology. Diagnosis Example : 1-Noninvasive diagnosis of liver cirrhosis. 2-To Confirm Fungemia Due to Trichosporon dermatis in a Pediatric Patient. Forensic Identify potential suspects whose DNA may match evidence left at crime scenes Exonerate persons wrongly accused of crimes Identify crime and catastrophe victims